Adeno-associated viruses (AAV) have unique features that make them attractive as vectors for gene therapy and genetics vaccines. AAV infection of cells in culture is noncytopathic, and natural infection of humans and other animals is silent, asymptomatic, and not implicated in the etiology of any human disease. Moreover, AAV infects a wide range of cell types including many mammalian cells, allowing the possibility of targeting many different tissues in vivo. AAV infects slowly dividing and non-dividing cells and can persist essentially for the lifetime of those cells as a transcriptionally active nuclear episome (extrachromosomal element). Integrated copies of rAAV vector in organs such as liver or muscle are very rare. Efficient long-term gene transfer has been reported in a number of cell types including eye, CNS, and muscle. See, e.g., X. Xiao et al., J. Virol. 70(11):8098-8108 (1996); R. R. Ali et al., Hum. Mol. Genet. 5(5):591-94 (1996). Current clinical studies have largely focused on the use of serotype 2 rAAV vectors, but a number of reports have demonstrated that other AAV serotypes including rAAV-1, rAAV-4, rAAV-5 and rAAV-8 have unique in vivo bio-distribution which make them attractive viral serotypes to test in clinical trials.
Adeno-associated virus (AAV) is a replication-deficient parvovirus, the single-stranded DNA genome of which is about 4.7 kb in length including 145 nucleotide inverted terminal repeat (ITRs). The nucleotide sequence of the AAV serotype 2 (AAV2) genome is presented in Srivastava et al., J. Virol., 45: 555-564 (1983) as corrected by Ruffing et al., J. Gen. Virol., 75: 3385-3392 (1994). Cis-acting sequences directing viral DNA replication (rep), encapsidation/packaging and host cell chromosome integration are contained within the ITRs. Three AAV promoters, p5, p19, and p40 (named for their relative map locations), drive the expression of the two AAV internal open reading frames encoding rep and cap genes. The two rep promoters (p5 and p19), coupled with the differential splicing of the single AAV intron at nucleotides 2107 and 2227, result in the production of four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene. Rep proteins possess multiple enzymatic properties which are ultimately responsible for replicating the viral genome. The cap gene is expressed from the p40 promoter and it encodes the three capsid proteins VP1, VP2, and VP3. Alternative splicing and non-consensus translational start sites are responsible for the production of the three related capsid proteins. A single consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158: 97-129 (1992).
AAV particles comprise a proteinaceous capsid having three capsid proteins, VP1, VP2 and VP3, which enclose a ˜4.6 kb linear single-stranded DNA genome. Individual particles package only one DNA molecule strand, but this may be either the plus or minus strand. Particles containing either strand are infectious, and replication occurs by conversion of the parental infecting single strand to a duplex form, and subsequent amplification, from which progeny single strands are displaced and packaged into capsids. Duplex or single-strand copies of AAV genomes (sometimes referred to as “proviral DNA” or “provirus”) can be inserted into bacterial plasmids or phagemids, and transfected into adenovirus-infected cells. See Carter, HANDBOOK OF PARVOVIRUSES, Vol. I, pp. 169-228 (1989), and Berns, VIROLOGY, pp. 1743-1764, Raven Press, (1990) for a general review of AAV.
rAAV vector production generally requires four common elements: 1) a permissive host cell for replication; 2) helper virus function which can be supplied by suitable helper viruses such as adenovirus or herpes virus, or alternatively by plasmid constructs containing the minimal adenoviral helper functions; 3) a trans-packaging rep-cap construct; and 4) a suitable production media.
Recombinant AAV particles can be produced from packaging cell lysates. See, e.g., Chirico and Trempe (1998) J. Virol. Methods 76:31-41. However, the cell lysate contains various cellular components such as host cell DNA, host cell proteins, media components and either helper virus or helper virus plasmid DNA which must be separated from the rAAV vector before it is suitable for in vivo use. Recent advances in rAAV production include the use of non-adherent cell suspension processes in stirred tank bioreactors and production conditions whereby rAAV vectors are released into the media or supernatant reducing the concentration of host cellular components present in the production material but still containing appreciable amounts of in-process impurities. See U.S. Pat. No. 6,566,118 and PCT WO 99/11764. Therefore, rAAV particles may be collected from the media and/or cell lysate and further purified.
Methods including density gradient centrifugation employed for the purification of rAAV vectors and in particular rAAV-2 are not amenable to scale up. Recent reports for rAAV-2 vectors have described purification methods employing ion exchange chromatography including opposing ion exchange chromatography (including cation and anion chromatography). See for example U.S. Pat. No. 6,566,118 and PCT WO 99/11764 which disclose methods of using a combination of opposing ion exchange chromatography for purifying recombinant adeno-associated virus vectors from a culture supernatant and/or a cell lysate. Additional improvements in rAAV stock preparations include the use of deoxycholate treatment of the cell lysate, iodixanol gradient separation prior to the affinity chromatography, which have resulted in high titer rAAV2 (Clark et al., Hum. Mol. Genet. 10(6):1031-39 (1999); Zolotukhin et al., Gene Therapy 6(6):973-985 (1999)). O'Riordan et al. (O'Riordan et al., J. Gene Med. 2:444-454 (2000); U.S. Pat. No. 7,015,026) also reported scalable chromatographic purification process for recombinant adeno-associated virus vectors and as particularly exemplified, rAAV-2 vectors, using ion exchange chromatography, hydroxyapatite chromatography, cellufine sulfate affinity chromatography, and zinc chelate chromatography.
Recent data indicate that rAAV capsid serotypes such as rAAV-1, 4, 5, and 8 bind weakly to anionic resins either as purified virus stock or in the presence of in-process production impurities such as host cell DNA, host cell proteins, serum albumin, media components, and helper virus components. Consequently, purification of those capsid serotypes typically involves anion-exchange chromatography in combination with other purification methods, such as iodixinol density-gradient centrifugation. See, e.g., Zolotukhin et al., Methods 28(2):158-167 (2002) and Kaludov et al., Hum. Gene Therapy 13:1235-1243 (2002); and U.S. Patent Publication No. 2004/0110266 A1. However, those methods are not readily scalable to commercial scale processes.
Accordingly, in the development of recombinant AAV vectors such as those for use in gene therapy and gene vaccines, there is a need for methods of purifying rAAV vectors from in-process production components including helper virus, as well as helper virus proteins, cellular proteins, host cell DNA, and media components present in the rAAV production stock. Such methods should be effectively employed on a scale that is suitable for the practical application of gene therapy techniques. Moreover there is a need for development of purification processes for rAAV vectors that are scalable to yield high titer, highly purified commercial stocks useful for rAAV gene therapy and gene vaccines. More particularly, there is a need for development of purification processes for rAAV vectors that bind weakly to chromatographic resins and in particular anionic resins.
The disclosures of all publications, patent applications, and patents cited in this specification are herein incorporated by reference as if each individual publication, patent application, or patent were specifically and individually indicated to be incorporated by reference. In particular, all publications cited herein are expressly incorporated herein by reference for the purpose of describing and disclosing compositions and methods which might be used in connection with the invention. Although the invention provided herein has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.